CAJ | 학술논문

目的：研究甲基强的松龙（MPSS）与促红细胞生成素（EPO）联合应用对体外培养的原代星形胶质细胞的作用。方法采用出生3 d内SD新生鼠的大脑制成细胞悬浊液后，以含15%胎牛血清的DMEM培养液培养，摇床后逐渐传代、纯化星形胶质细胞，磷酸盐缓冲液（PBS）代替培养基模拟缺营养模型3 h后即刻分别加入MPSS 10μmol/L，EPO 10 U/L，MPSS 10μmol/L﹢EPO 10 U/L，分别继续培养3 d，采用免疫荧光技术鉴定星形胶质细胞，MTT法检测细胞的增殖活性和凋亡，聚合酶链式反应（PCR）技术测定细胞胶质纤维酸性蛋白( GFAP ) mRNA表达水平。结果星形胶质细胞摇床后传至第3代，经鉴定细胞纯度达95%以上；缺营养3 h后部分细胞形态变圆甚至死亡漂浮于正常细胞表面；正常组和损伤组分别加入MPSS，EPO，MPSS﹢EPO 3 d后，损伤组细胞活性与GFAP mRNA表达水平均明显升高（ P﹤0.05），且MPSS和EPO联合应用与单用MPSS、单用EPO相比，细胞活性与GFAP mRNA表达水平明显升高（ P﹤0.05），但正常组无明显变化（ P﹥0.05）。结论适当剂量的MPSS与EPO联合应用对缺营养损伤星形胶质细胞有显著的保护作用，且与单用MPSS、单用EPO相比有显著性差异，但对正常星形胶质细胞无显著的保护作用。
목적：연구갑기강적송룡（MPSS）여촉홍세포생성소（EPO）연합응용대체외배양적원대성형효질세포적작용。방법채용출생3 d내SD신생서적대뇌제성세포현탁액후，이함15%태우혈청적DMEM배양액배양，요상후축점전대、순화성형효질세포，린산염완충액（PBS）대체배양기모의결영양모형3 h후즉각분별가입MPSS 10μmol/L，EPO 10 U/L，MPSS 10μmol/L﹢EPO 10 U/L，분별계속배양3 d，채용면역형광기술감정성형효질세포，MTT법검측세포적증식활성화조망，취합매련식반응（PCR）기술측정세포효질섬유산성단백( GFAP ) mRNA표체수평。결과성형효질세포요상후전지제3대，경감정세포순도체95%이상；결영양3 h후부분세포형태변원심지사망표부우정상세포표면；정상조화손상조분별가입MPSS，EPO，MPSS﹢EPO 3 d후，손상조세포활성여GFAP mRNA표체수평균명현승고（ P﹤0.05），차MPSS화EPO연합응용여단용MPSS、단용EPO상비，세포활성여GFAP mRNA표체수평명현승고（ P﹤0.05），단정상조무명현변화（ P﹥0.05）。결론괄당제량적MPSS여EPO연합응용대결영양손상성형효질세포유현저적보호작용，차여단용MPSS、단용EPO상비유현저성차이，단대정상성형효질세포무현저적보호작용。
Objective To study the effect of methylprednisolone(MPSS) combined with erythropoietin(EPO) to in vitro cultured primary astrocytes(AST). Methods The cell suspension was prepared by using the cerebrum of neonatal rats within 3 d of birth,cultured by the DMEM culture solution containing 15% fetal calf serum,gradually passaged after shaking for purifying astrocytes. After simulating nulli-nutrition model for 3 h by phosphate buffer solution(PBS) for substituting medium,MPSS 10 μmol/L,EPO 10 U/L and MPSS 10 U/L plus EPO 10 U/L were immediately added and the continuous culture for 3 d was performed. Astrocytes were identified by the immunofluorescent technique;the proliferation activity and apoptosis of astrocytes were detected by MTT;the expression level of GFAP mRNA was detected by PCR. Results Astrocytes were passaged to the third generation after shaking,the astrocytes purity reached more than 95% by identification;part of astrocytes became round in morphology and even died after nulli-nutrition for 3 h;after 3 d of applying MPSS,EPO and MPSS plus EPO in the normal group and the damage group,both cytoactive and expression level of GFAP mRNA in the damage group were significantly increased ( P ﹤ 0. 05 ) ,moreover compare with the single application of MPSS or EPO,the cytoactive and GFAP mRNA expression level in the combined application of MPSS and EPO were significantly increased( P ﹤ 0. 05),but no obvious change was found in the normal group( P ﹥ 0. 05). Conclusion The combined application of proper dose of MPSS and EPO has obviously protection effect on nulli-nutrition damaged astrocytes,moreover has significant difference compared with the single appli-cation of MPSS or EPO,but has no obvious protective effect on normal astrocytes.